阅读说明：本技术 一种从蜂蜜中提取活性物质的深加工萃取方法 (Deep processing extraction method for extracting active substances from honey ) 是由 肖维刚 于 2019-01-23 设计创作，主要内容包括：本发明公开了一种从蜂蜜中提取活性物质的深加工萃取方法,包括活性生物酶与多糖的分离、活性生物酶提纯、多糖提纯和活性物质保存使用,活性生物酶与多糖的分离,通过盐溶液提取法进行分离提纯,在蜂蜜中添加缓冲液,充分混合搅拌,控制中性盐浓度和混合环境,混合后溶液使用盐析法提取活性酶混合物,在蜂蜜的混合溶液中继续添加中性盐,盐析的过程中将溶液的PH设定在各个活性酶的等电点。本发明使用碱性磷酸盐使得蜂蜜中的活性生物酶与中性盐结合,从而使得活性生物酶与多糖相互之间明显的分离开来,便于后期的对两者分别进行提纯,操作环境控制在低温,中性偏碱性环境中,能够有效的避免其中活性生物酶的失活,以及避免蛋白质的水解。(The invention discloses a deep processing extraction method for extracting active substances from honey, which comprises the steps of separating active biological enzyme from polysaccharide, purifying the active biological enzyme, purifying the polysaccharide and storing the active substances, separating the active biological enzyme from the polysaccharide, separating and purifying the polysaccharide by a salt solution extraction method, adding a buffer solution into the honey, fully mixing and stirring, controlling the concentration of neutral salt and the mixing environment, extracting an active enzyme mixture by using a salting-out method for the mixed solution, continuously adding the neutral salt into the mixed solution of the honey, and setting the pH of the solution at the isoelectric point of each active enzyme in the salting-out process. The invention combines the active biological enzyme and the neutral salt in the honey by using the alkaline phosphate, thereby obviously separating the active biological enzyme and the polysaccharide, facilitating the purification of the active biological enzyme and the polysaccharide respectively at the later stage, effectively avoiding the inactivation of the active biological enzyme and avoiding the hydrolysis of protein by controlling the operating environment at low temperature and in the neutral alkaline environment.)

1. A deep processing extraction method for extracting active substances from honey is characterized by comprising the following steps: separating active biological enzyme from polysaccharide, purifying active biological enzyme, purifying polysaccharide and storing active substances for use;

s1, separation of active biological enzyme and polysaccharide: separating and purifying by salt solution extraction method, adding buffer solution into Mel, mixing and stirring, and controlling neutral salt concentration and mixing environment;

s2, extracting the active enzyme mixture from the mixed solution by using a salting-out method, continuously adding neutral salt into the mixed solution of the honey, and setting the pH value of the solution at the isoelectric point of each active enzyme in the salting-out process;

s3, separating out protein precipitate, and filtering solid and liquid to separate out solid precipitate and residual honey solution;

s4, purification of active biological enzyme: the solid precipitate is dialyzed, the precipitated precipitate and the buffer solution are mixed into solution again, a dialysis bag is used for separating macromolecular active biological enzyme and neutral salt solution, and the substance retained in the dialysis bag is active biological enzyme;

s5, polysaccharide purification: removing impurities from the residual solution of Mel by membrane separation method, separating with fiber filter and semipermeable membrane, outputting mixed water solution of neutral salt and small molecular impurities, and retaining on the semipermeable membrane in the fiber filter to obtain polysaccharide mixture.

2. A further process for the extraction of an active substance from honey as claimed in claim 1, wherein the separation environment of the active biological enzyme from the polysaccharide is controlled at 0 ℃ to 5 ℃, the temperature is controlled in a sterile dust-free cabinet, the extraction buffer is 0.02 to 0.05M isotonic saline solution of phosphate, a small amount of neutral salt such as NaCl is added during the extraction process, typically at a concentration of 0.15 mol/L, at an ambient pH of above 7.0, and the mixture is thoroughly mixed with stirring.

3. A further process for the extraction of an active substance from honey as claimed in claim 1, wherein the neutral salt used in the separation of the active bio-enzyme from the polysaccharide is ammonium phosphate (NH)₄)₃PO₄The solubility of ammonium phosphate at 20 ℃ is 28.3g/100ml, the pH is controlled to 6.0 or more, and the pH of the solution is set at the isoelectric point of each active enzyme during salting out.

4. A further process for the extraction of an active substance from honey as claimed in claim 1, wherein the buffer solution in the purification of active bio-enzyme is 0.02-0.05M isotonic saline solution of phosphate, the purification of active bio-enzyme uses a separation drum, the separated precipitate and the buffer solution are mixed again to form a solution, the separation drum contains water, the separation drum is equipped with an internal dialysis bag, the mixed solution is filled into the internal dialysis bag, the solution in the internal dialysis bag is slowly stirred at variable speed, and the dialysis is carried out while stirring with a stirring paddle.

5. A further process for the extraction of an active substance from honey as claimed in claim 1, wherein the semipermeable membrane is installed in the fibrous filter, the residual honey solution is gradually introduced into the fibrous filter, and the residual honey solution is filtered through the semipermeable membrane, and the polysaccharide mixture retained in the filter is scraped off the semipermeable membrane.

6. A further process for extracting active substances from honey as claimed in claim 1, wherein the active bio-enzyme is preserved, the active bio-enzyme is prepared into lyophilized powder or is preserved in crystal state, the preservation time is up to 8 years at-15 deg.C, and the polysaccharide mixture can be directly used for production.

Technical Field

The invention relates to the technical field of honey processing, in particular to a deep processing and extracting method for extracting active substances from honey.

Background

The honey is a food which is brewed in a honeycomb and stored for eating in the future after the honey is collected by bees, plant nectar or nectar and honeydew are added with secretion of the digestive tract of the bees, the honey contains 180 different substances, the main components of the honey are saccharides, including monosaccharide, disaccharide, oligosaccharide, polysaccharide and the like, glucose and fructose in the monosaccharide account for 85% -95% of total sugar, and in addition, the honey also contains various organic acids, amino acids, cane sugar, vitamins, mineral substances, ferment, aromatic substances, pigments, hormones, enzymes, bioactive substances and the like, so the nutrition is very rich.

The honey has high nutritive value, contains a large amount of bioactive substances required by human beings, can be absorbed by a direct eating method generally, the bioactive substances in the honey are sensitive to temperature, meanwhile, most of the honey used in the modern process is not brewed honey, the internal bioactive substances are easy to inactivate and cause loss after multiple processing, and the most valuable substances in the honey are the bioactive substances.

The active biological enzyme in the honey is used as a natural catalyst, can decompose polysaccharide in the honey into monosaccharide which can be directly absorbed by people, cannot be artificially synthesized at present, and is beneficial to the maturity of the honey, but most of the existing honey preparation methods simply reduce the water content in raw honey and cannot catalyze the maturity of the honey.

Disclosure of Invention

The invention aims to solve the defects in the prior art, such as: most of honey used in the modern process is not brewed honey, internal bioactive substances are easy to inactivate after a plurality of times of processing, loss is caused, the content of active biological enzyme is beneficial to the maturity of the honey, but most of the existing honey preparation is only simple to reduce the water content in the raw honey and can not catalyze the maturity of the honey, and the deep processing extraction method for extracting the active substances from the honey is provided.

In order to achieve the purpose, the invention adopts the following technical scheme:

a deep processing extraction method for extracting active substances from honey comprises the following steps: separating active biological enzyme from polysaccharide, purifying active biological enzyme, purifying polysaccharide and storing active substances for use;

s1, separation of active biological enzyme and polysaccharide: separating and purifying by salt solution extraction method, adding buffer solution into Mel, mixing and stirring, and controlling neutral salt concentration and mixing environment;

s2, extracting the active enzyme mixture from the mixed solution by using a salting-out method, continuously adding neutral salt into the mixed solution of the honey, and setting the pH value of the solution at the isoelectric point of each active enzyme in the salting-out process;

s3, separating out protein precipitate, and filtering solid and liquid to separate out solid precipitate and residual honey solution;

s4, purification of active biological enzyme: the solid precipitate is dialyzed, the precipitated precipitate and the buffer solution are mixed into solution again, a dialysis bag is used for separating macromolecular active biological enzyme and neutral salt solution, and the substance retained in the dialysis bag is active biological enzyme;

s5, polysaccharide purification: removing impurities from the residual solution of Mel by membrane separation method, separating with fiber filter and semipermeable membrane, outputting mixed water solution of neutral salt and small molecular impurities, and retaining on the semipermeable membrane in the fiber filter to obtain polysaccharide mixture.

Preferably, the separation environment is controlled in the separation of the active biological enzyme and the polysaccharide, the temperature is 0-5 ℃, the sterile dust-free operation box is adopted, the extraction buffer solution is 0.02-0.05M phosphate isotonic salt solution, a small amount of NaCl and other neutral salt is added in the extraction process, the concentration is preferably 0.15 mol/L, the pH value of the environment is neutral and is above 7.0, and the components are fully stirred and mixed.

Preferably, the neutral salt used in the separation of the active biological enzyme from the polysaccharide is ammonium phosphate (NH4)₃PO₄The solubility of ammonium phosphate at 20 ℃ is 28.3g/100ml, the pH is controlled to 6.0 or more, and the pH of the solution is set at the isoelectric point of each active enzyme during salting out.

Preferably, the buffer solution in the purification of the active biological enzyme is 0.02-0.05M phosphate isotonic salt solution, the separation cylinder is used for the purification of the active biological enzyme, the precipitated precipitate and the buffer solution are mixed into solution again, water is filled in the separation cylinder, a built-in dialysis bag is installed in the separation cylinder, the mixed solution is filled in the built-in dialysis bag, the solution in the built-in dialysis bag is slowly stirred at variable speed, and the dialysis is carried out while stirring by using a stirring paddle.

Preferably, the semipermeable membrane is arranged in the fiber filter during the purification of the polysaccharide, the residual honey solution is gradually input into the fiber filter, the honey solution is filtered by the inner semipermeable membrane, and the polysaccharide mixture remained in the honey solution is scraped from the semipermeable membrane.

Preferably, the extracted active biological enzyme needs to be stored, the active biological enzyme is prepared into lyophilized powder or stored in a crystal state, the active biological enzyme is stored in a low-temperature freezing environment at the temperature of-15 ℃, the storage time reaches 8 years, and the polysaccharide mixture can be directly used for production and use.

Compared with the prior art, the invention has the beneficial effects that:

1. the active biological enzyme in the honey is combined with neutral salt by using the alkaline phosphate, so that the active biological enzyme and the polysaccharide are obviously separated from each other, the active biological enzyme and the polysaccharide are conveniently purified respectively in the later period, the operation environment is controlled in a low-temperature neutral alkaline environment, the inactivation of the active biological enzyme in the honey can be effectively avoided, and the hydrolysis of protein in an acid-too-acid or alkali-too environment is avoided.

2. By salting out, using the limit of the solubility of neutral salt in water, continuously adding neutral salt to separate out the active biological enzyme combined with neutral salt from the solution, and separating the separated out fixed precipitate from the solution by simple solid-liquid filtration, the active biological enzyme is separated from honey and separated by converting into two different physical states.

3. And (3) mixing the precipitated precipitate and the buffer solution into a solution again by using a dialysis method, using a dialysis bag, wherein the substance retained in the dialysis bag is active biological enzyme, the permeated substance is neutral salt, the protein molecules of the active biological enzyme are far larger than the neutral salt, and purifying by using the size of the molecules.

4. Purifying the solution containing polysaccharide by membrane filtration, filtering out small molecular impurities and neutral salt in the solution by using a fiber filter, and reserving the polysaccharide mixture, wherein the polysaccharide substance can be directly put into production and use.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the illustrated orientations or positional relationships for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.

A deep processing extraction method for extracting active substances from honey comprises the following steps: separating active biological enzyme from polysaccharide, purifying active biological enzyme, purifying polysaccharide and storing active substances for use;

s1, separating and purifying the active biological enzyme and the polysaccharide, namely, adding a buffer solution into honey, fully mixing and stirring, controlling the concentration of neutral salt and the mixing environment, controlling the separation environment in the separation of the active biological enzyme and the polysaccharide, controlling the temperature to be 0-5 ℃, using a sterile dust-free operation box, extracting the buffer solution into a 0.02-0.05M salt penetrating solution of phosphate and the like, adding a small amount of neutral salt such as NaCl and the like in the extraction process, generally preferably 0.15 mol/L concentration, and fully stirring and mixing when the pH value of the environment is neutral and is more than 7.0;

s2, extracting the mixture of active enzymes with salting-out method, adding neutral salt into the mixed solution of Mel, setting pH of the solution at isoelectric point of each active enzyme during salting-out process, and separating active biological enzyme from polysaccharide with neutral salt of ammonium phosphate (NH4)₃PO₄The solubility of ammonium phosphate at 20 ℃ is 28.3g/100ml, the pH value is controlled to be more than 6.0, and the pH value of the solution is set at the isoelectric point of each active enzyme in the salting-out process;

s3, separating out protein precipitate, and filtering solid and liquid to separate out solid precipitate and residual honey solution;

s4, purification of active biological enzyme: the method comprises the following steps of (1) carrying out dialysis on solid precipitates, mixing the precipitated precipitates with a buffer solution to obtain a solution again, separating macromolecular active biological enzyme and a neutral salt solution by using a dialysis bag, mixing the substances retained in the dialysis bag to obtain an active biological enzyme, purifying the active biological enzyme, wherein the buffer solution is a 0.02-0.05M isotonic phosphate solution, purifying the active biological enzyme by using a separation cylinder, mixing the precipitated precipitates with the buffer solution to obtain a solution again, adding water into the separation cylinder, installing an internal dialysis bag in the separation cylinder, filling the mixed solution into the internal dialysis bag, slowly stirring the solution in the internal dialysis bag at a variable speed, and carrying out dialysis while stirring by using a stirring paddle;

s5, polysaccharide purification: impurity is got rid of to the remaining solution of honey using the membrane separation method, use fibrous filter and pellicle to separate, the output be neutral salt and small molecule impurity's mixed water solution, keep somewhere in the fibrous filter on the pellicle be the polysaccharide mixture, the pellicle is installed in the fibrous filter in the polysaccharide purification, in importing the fibrous filter gradually with the remaining solution of honey, filter through inside pellicle, keep somewhere that the polysaccharide mixture in inside scrapes from the pellicle and get off can.

The extracted active biological enzyme needs to be stored, the active biological enzyme is prepared into freeze-dried powder or stored in a crystal state, the active biological enzyme is stored in a low-temperature freezing environment at the temperature of-15 ℃, the storage time reaches 8 years, and the polysaccharide mixture can be directly used for production and use.

Most of bioactive substances in honey are sugars and other bioactive substances, enzymes contained in the honey are special proteins and have extremely strong bioactivity, the quantity of the enzymes contained in the honey, namely the enzyme value is an important index for testing the maturity of the honey, the enzymes in the honey are mainly added by bees in the honey brewing process, the sources of the enzymes are the saliva of the bees, mainly sugarcane enzymes, the enzymes can convert sucrose in nectar into glucose and fructose, and in addition, amylase, glucose oxidase, catalase, reductase, invertase, phosphatase, protease-like enzyme and the like are added, and the biological enzymes in the honey are extracted and extracted, so that the biological enzymes can be directly utilized, the nutritional value of the honey can be increased, and the conversion rate of the sugars in the honey can be improved.

Firstly, the collected raw honey is purified and extracted, the extracted biological enzyme needs to ensure the biological activity and needs to be controlled by the environment, the temperature of the extracted liquid needs to be controlled between 0 ℃ and 5 ℃, the pH value of the liquid is 7.0, and the extraction environment needs to adopt a sterile dust-free operation box, so that the impurities such as dust, bacteria and the like in the environment are prevented from entering the honey in the operation process because the honey is viscous liquid.

Enzyme in honey is extracted by a salt solution extraction method, protein and enzyme are ampholytes with isoelectric points, the enzyme is inactivated under the environment of pH5.6, so a slightly alkaline solution is used for extraction, the activity of the active enzyme is ensured in the extraction process, the environment is kept below 5 ℃ and above 0 ℃, the freezing of an extracting solution is avoided while the enzyme is inactivated, a small amount of neutral salt such as NaCl is added into the extracting solution, the concentration is preferably 0.15 mol/L, a buffer solution is usually 0.02-0.05M of phosphate isotonic salt solution, and dilute salt solution ions are used for being combined with the active enzyme, namely the part of the protein, so that the proteins such as the active enzyme in the honey can be obviously distinguished from polysaccharide.

Neutral salt is combined with active enzyme in the extract, the neutral salt has obvious influence on the solubility of protein, generally, the enzyme solubility is increased along with the increase of the salt concentration under the condition of low salt concentration, the salt solution, namely the extract concentration, is decreased in different degrees in the increasing process of the active enzyme solubility and is separated out in sequence, a large amount of salt is added into the active enzyme solution, high-concentration salt particles have strong hydration power and can capture a hydration layer in the active enzyme, then the active enzyme coagulates, precipitates and separates out, the pH of the solution is set at the isoelectric point of each active enzyme in the salting-out process to be beneficial to the separation of the active enzyme, and the neutral salt adopts ammonium phosphate (NH4)₃PO₄The solubility at 20 ℃ was 28.3g/100ml, and the pH was controlled to 6.0 or more.

The precipitated precipitate is the combination of protein of active enzyme and neutral salt, the salt is removed, the precipitated precipitate and buffer solution are mixed into solution by dialysis method, the solution is filled into dialysis bag, dialysis is carried out while stirring, finally macromolecule active biological enzyme is retained in the dialysis bag, and the operation environment is kept at low temperature.

So far, the macromolecular active enzyme in the honey is separated and extracted, the remaining solution is polysaccharide solution, and the remaining solution contains other micromolecular impurities which need to be removed, and the extracted high-purity polysaccharide can be directly put into food production.

The existence of micromolecule impurities can influence the biological activity of polysaccharide, further separation is needed, the purity is improved, the remaining solution contains water, the viscosity of corresponding honey is greatly reduced, a fiber filter is used for purifying the polysaccharide in the water solution by adopting a membrane separation method, micromolecule substances can permeate through the polysaccharide, but the polysaccharide is macromolecular substances and cannot permeate through the polysaccharide, so that the remaining polysaccharide with high purity is obtained, the whole purification production period is short, and the method is simple and direct.

The active biological enzyme which can be used for many times in the honey is extracted and then is prepared into freeze-dried powder or is stored in a crystal state, the active biological enzyme is stored at low temperature of-15 ℃, the extracted and stored active biological enzyme can be used in the honey collected next time, the process of bee brewing is simulated, the active biological enzyme is gradually increased, then, the water content is more in artificial environment, the insufficient decomposed raw honey is prepared into mature raw honey, the mature raw honey can be directly put into use, the active biological enzyme and polysaccharide can also be purified and separated, and the separated polysaccharide can be directly put into production and use.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.